Horseshoe crab has a type of hemocytes in the body and the hemocytes are filled with two kinds of large and small granules having different densities. The large granules contain a body fluid coagulation factor and an anti-lipopolysaccharide factor, and the small granules contain antimicrobial substances such as tachyplesin. The inventors of the present invention isolated six proteins, S1-S6, from hemocytes of horseshoe crab as components of small granules (Shigenaga, T. et al., J. Biochem., 114, 307-316 (1993)).
In addition, a group of peptides having antimicrobial activity and generally called defensins were isolated from cellular granules of neutrophils and macrophages of mammals such as human being, rabbits, guinea pigs and rats, and their total amino acid sequences are disclosed in U.S. Pat. No. 5,242,902. The growth stimulant effect on epidermal cells and fibroblast cells of defensins were disclosed in said U.S. Pat. No. 5,242,902 in addition to the antimicrobial activity against Gram negative and positive bacteria and fungi. Defensins are basic peptides having about 30 amino acid residues with common components of six cysteine residues and three disulfide bonds.
No polypeptide isolated from the hemocytes of horseshoe crab having similar amino acid sequence with those of defensins has been reported. Furthermore, no nucleotide sequence corresponding to the amino acid sequence of polypeptide of the present invention has been known.
The inventors of the present invention further investigated the S5 peptide fraction in the isolated components of small granule fractions of hemocytes of horseshoe crab and determined its total amino acid sequence. The amino acid sequence has a similar structure in the C-terminal with that of known defensins. The antimicrobial activity of S5 peptide fraction was further investigated and S5 peptide fraction was found to have potent antimicrobial activities against Gram positive and negative bacteria and fungi, and accomplished the present invention.